Method of detecting fluorescent materials and apparatus for their detection

ABSTRACT

A method for detecting fluorescent materials in liquid chromatography. Fluorescent materials are excited by chemical reaction with chemical reagents to yield light (chemiluminescence) and detected at high sensitivity. The detection apparatus is composed of a mixer for mixing a solution containing separated fluorescent materials and solutions of chemical reagents to afford chemiluminescence, a flow cell and a light receptive detection means for detecting the light thus produced.

This application is a continuation of application Ser. No. 292,487,filed Aug. 31, 1981 which is a division of application Ser. No. 80,294,filed Oct. 1, 1979, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of detecting fluorescent materialsand also an apparatus for their detection. More particularly, theinvention relates to a method and apparatus for detecting fluorescentmaterials at high sensitivity in liquid chromatography by excitingfluorescent materials to produce light by chemical reaction withchemical reagents.

2. Description of the Prior Art

Liquid chromatography is widely used for separating and quantitativelyanalyzing one or several kinds of materials in a mixture of variouscomponents. When the material or materials to be separated andquantitatively analyzed have absorptions in the ultraviolet or visiblewavelength region, an absorptions spectrophotometer is usually used asthe detection means in liquid chromatography. However, when a highersensitivity is required, for example, in the case where the amount ofthe aforesaid material or materials to be analyzed in the sample mixtureis very small, a fluorescence detector is employed as the detectionmeans in liquid chromatography.

A conventional fluorescence detector for liquid chromatography iscomposed of a flow cell through which a solution containing separatedfluorescent material is passed, a light source for exciting thefluorescent material, and a light-receptive detection means forperceiving and multiplying the light (fluorescence) generated(Instrumental liquid chromatography p. 81, ed by N. A. Paris, ElsevierScientific Publishing Comp. 1976). In the conventional fluorescencedetector, undesirable phenomena, such as the stray light phenomenonwherein a part of light from the light source, for exciting thefluorescent materials, enters the light-receptive detector and adeviation of the light from the light source induces a deviation in thefluorescence. Thus, an attempt to increase the sensitivity of thedetector leads to, at the same time, an increase in the noise level andit is difficult to increase the signal to noise ratio (S/N). In such afluorescent detector, there is a limitation in the increment of thedetection sensitivity and thus the detector is insufficient for thedetection of a very minute amount of fluorescent materials.

On the other hand, a means for increasing the sensitivity, has beendeveloped in which without using a flow cell for reducing stray light,special light (strong laser light is applied onto a droplet of theeluate from the column for liquid chromatography to excite thefluorescent materials, but the precision is low and the cost of theapparatus is very high. Therefore, the development of a method whereinfluorescent materials can be detected with high sensitivity and highprecision using an inexpensive apparatus has been desired.

SUMMARY OF THE INVENTION

Accordingly, on noticing the principle that fluorescent materials can bechemically excited to give light (chemiluminescence) with chemicalreagents, the inventors have tried to apply the aforesaid principle to adetection means for liquid chromatography and investigated in detail theselection and the mixing ratio of solvents used for the chemicalreaction for chemiluminescence, the concentration of an oxalic acidderivative and a peroxide which are chemical reagents, an the influenceof catalyst using dansylamino acids as representatives for fluorescentmaterials. As the result thereof it has been found that a very minuteamount of a fluorescent material can be quantitatively analyzed bymixing a solution containing separated fluorescent materials andsolutions of chemical reagents to cause chemiluminescence and detectingthe light. Furthermore, as the result of earnest investigations forfurther increasing the detection sensitivity, the detecting apparatus asshown schematically in FIG. 1 of the accompanying drawings has beendeveloped and the invention has been accomplished.

According to this invention there is provided a method, for liquidchromatography, of detecting fluorescent materials which are chemicallyexcited to give light with chemical reagents.

Also, according to another embodiment of this invention, there isprovided an apparatus for detecting fluorescent materials having nolight source in a liquid chromatographic means, comprising a mixer formixing a solution containing separated fluorescent material or materialsand solutions of chemical reagents for chemiluminescence, a mixing coilif necessary, a flow cell through which the above mixed solution passesand a light receptive detection means for detecting the light producedby the chemical reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram showing schematically the detection method andapparatus of this invention,

FIG. 2 is a chromatogram obtained by applying this invention to astandard mixed solution of 3 kinds of 4×10⁻¹³ mole dansylamino acids,and

FIG. 3 shows calibration curves for the dansylamino acids aforesaid ineach injection amount.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "fluorescent material" which is quantitatively detected in thisinvention means not only a fluorophore, i.e., a material which emitsfluorescent light on irradiation with exciting light (visible light orultraviolet light) but also a fluorescent derivative which can be madeto emit fluorescent light, which is formed by the reaction of anon-fluorescent material with some reagent. Practical examples offluorophores are polycyclic aromatic hydrocarbons such as benzpyrene,etc.; biogenic amines; vitamins such as vitamin B₂, vitamin B₁₂, vitaminE, etc,; and medicaments such as salicylic acid, etc. Fluorescentderivatives, are materials obtained by reacting non-fluorescentmaterials such as aminoacids, peptides, proteins, biogenic amines (forexample, catecholamines), etc., with a fluorescent reagent such asdansyl chloride, fluorescamine, o-phthalaldehyde, etc.

Therefore, when the material separated and quantitatively determined inthis invention is a fluorophore, the material is separated as it is byliquid chromatography and then quantitatively determined by thedetection apparatus. On the other hand, when the material is anon-fluorescent material, the material can be converted into afluorescent derivative prior to the separation by liquid chromatographyand quantitatively analyzed after being separated by liquidchromatography, or the material can be subjected to a derivatizationreaction after being separated by liquid chromatography to form afluorescent derivative and then quantitatively determined. Accordingly,the term "a solution containing separated fluorescent materals" in thisinvention means both the column eluate containing the aforesaidfluorophore or fluorophores, or the fluorescent derivative orderivatives and the solution of the fluorescent derivative orderivatives obtained by converting the non-fluorescent material ormaterials separated by column into fluorescent derivative orderivatives.

In addition, as a means for exciting a fluorescent material, a method bychemical reaction without need of light irradiation is known as shownbelow (see, M. Rauhut et al; "J. Am. Chem. Soc." 89, 6515(1967)):##STR1##

Also, a method wherein both a solution of an oxalic acid ester and asolution of hydrogen peroxide are sprayed on a thin-layer chromatogramto yield chemiluminescence from fluorescent material separated bythin-layer chromatography and a method wherein hydrogen peroxide isquantitatively analyzed in the presence of oxalic acid ester and a largeexcess of fluorescent material are known (see, T. G. Curtis et al; "J.Chromatogr.", 134, 343(1977) and D. C. Williams et al; "Anal. Chem.",48, 1003(1976).

However, the chemical reaction for chemiluminescence has never beenutilized for the quantitative detection of a fluorescent material usingliquid chromatography. By use of the detection method of this invention,a fluorescent material can be separated and quantitatively determinedwith higher detection sensitivity compared with that in a conventionaldetection method for fluorescent material. For example, dansyl aminoacids can be detected with about 10³ times more sensitivity than thatobtainable in a conventional method. The detection method and thedetection apparatus of this invention will be explained in more detailby referring to the accompanying drawings.

FIG. 1 is an example of block diagram showing schematically thedetection apparatus of this invention. That is, as is clear from FIG. 1,the apparatus of this invention is equipped with a detection systemcomprising

(1) a section for mixing a solution containing the separated fluorescentmaterial or materials and solutions of chemical reagents forchemiluminescence and

(2) a section for detecting the light produced by the chemical reaction(that is, a flow cell and a light receptive detection means forperceiving and multiplying the light produced by the chemical reactionfor chemiluminescence)

in place of a conventional detection system for liquid chromatography.The solutions of chemical reagents for chemiluminescence are sent to theaforesaid mixing section (FIG. 1) by means of a pump at a constant rateand mixed with the solution containing the separated fluorescentmaterial or materials.

Since the chemiluminescence is influenced by the variance in compositionof the reaction solutions, it is necessary in the detection apparatus ofthis invention to keep the ratio of the three solutions, that is, asolution containing the separated fluorescent material or materials, asolution of an oxalic acid derivative, and a solution of peroxide,passing throught a flow cell at a constant value and for the purpose itis desirable to use a syringe type pump giving no pulsating current fortransferring each solution. However, when a reciprocating pump is usedfor reasons of pressure resistance, etc., the detection apparatus ofthis invention may be practiced by providing a means of eliminating thepulsating current.

In order to obtain emitted light with good reproducible intensity, it isnecessary to perform the reaction for chemiluminescence under theconditions wherein each solute and solution are mixed uniformly. Forthis reason, in the detection apparatus of this invention, it isprefered to use a mixer for promoting the mixing of the aforesaid threekinds of solutions at the zone where the solutions join, and furtherwhen mixing of the solutions is insufficient by the use of such a mixer,it is preferable to use a mixing coil having a sufficient length touniformly mix the solutions before they reach the flow cell. Inaddition, in the embodiment shown in FIG. 1, a solution containing theseparated fluorescent material or materials and two kinds of solutionsof chemical reagents for chemiluminescence are simultaneously introducedinto a mixer, but pre-mixed solutions of the chemical reagents may beintroduced into the mixer through a single conduit-line.

Since in the detection apparatus of this invention a light source forexciting fluorescent material is unnecessary, the influence of the lightsource on the sensitivity in a conventional fluorescence detector isremoved and it is possible to allow a flow cell to come as close aspossible to the light-receptive detection means and detect the lightemitted from the fluorescent material over the whole spectrum ofwavelengths. Furthermore, the flow cell and the light-receptivedetection means which are used conventionally can be employed in thisinvention but it is preferred that they be so constructed that the lightgenerated by the chemical reaction for chemiluminescence can beeffectively detected.

An oxalic acid derivative which is one component of the chemicalreagents for chemiluminescence used in this invention is a materialwhich brings a fluorescent material into an excited state to give lightunder the reaction with another component, i.e., a peroxide. Practicalexamples of the oxalic acid derivatives are an oxalic acid ester, anoxalic acid chloride, an oxalic acid anhydride, oxalic acid amide, etc.,(see, M. M. Rauhut et al; "J. Am. Chem. Soc." 89, 6515(1967): ibid., 88,3604(1966); I. J. Bollyky et al: ibid., 89, 6523(1967); and M. M.Rauhut; "Acc. Chem. Res." 2, 80(1969)). Also, any peroxides (that is,organic peroxide such as performic acid, peracetic acid, perpropionicacid, perbenzoic acid, etc., or inorganic peroxide such as hydrogenperoxide, persulfuric acid, etc.) may be used as the other component buton considering the efficiency for chemiluminescence, etc., hydrogenperoxide is most suitable.

Also, the solvent for dissolving the oxalic acid derivative should beselected on considering the kind and solubility of the oxalic acidderivative used, the stability of the reagent solution, the efficiencyfor chemiluminescence, etc., but in particular methyl acetate or ethylacetate is preferred. On the other hand, as a solvent used for theperoxide solution, the same solvent as for the oxalic acid derivative isused when the eluting solvent for liquid chromatography is easily mixedwith the solvent for the oxalic acid derivative. However, when anaqueous buffer solution is used as the eluting solvent, an organicsolvent which can be uniformly mixed with water, such as methanol,ethanol, acetone, acetonitrile, etc., is selectively used in order toaid the uniform mixing of the solvents.

The conditions such as the mixing ratio of solutions, etc., are properlyselected on considering the solvents used and the efficiency forchemiluminescence.

An example of separating and quantitatively determining a mixed sampleof 3 kinds of dansylamino acids by the detection method of thisinvention is shown in FIG. 2.

The analysis of dansylamino acids has been performed by liquidchromatography equipped with a conventional fluorescence detector but upto the detection limit reported required an amount of dansylamino acidsreported to be about 3×10⁻¹¹ mole (Hiroshi Wada et al; "Kagaku noRyoiki", an extra supplement, 114, 1(1976)) and in an ordinary analysis,it is said that dansylamino acid of the order of 10⁻¹⁰ to 10⁻⁹ mole isnecessary (T. Seki et al; "J. Chromatogr.", 102, 251(1974)).

On the other hand, as is clear from the example shown below, in thedetection apparatus of this invention the calibration curve for eachdansylamino acid shows good linearity to 5×10⁻¹⁴ mole from 8×10⁻¹³ moleas the amount injected into liquid chromatography and hence thedetection limit is 10⁻¹⁴ mole (S/N≈2).

Therefore, according to the detection method of this invention,fluorescent materials such as dansylamino acids can be detected with agood precision at a sensitivity of about 10³ times higher than that in aconventional detection method for fluorescent materials by properlyselecting the packing for a column and the suitable conditions forchemiluminescence. Thus, almost all fluorescent materials are expectedto be detected at a substantially higher sensitivity than that in aconventional detection method.

EXAMPLE

A sample prepared by mixing 4×10⁻¹³ mole of dansyl-glutamic acid,4×10⁻¹³ mole of dansyl-alanine and 4×10⁻¹³ mole of dansyl-methionine wassupplied to a column packed with μ-Bonda Pack C-18 (a trade name, madeby Waters Co.) and the dansylamino acids were separated and detected.The eluting solvent was 38% acetonitrile-0.05Mtrishydroxymethylaminomethane-hydrochloric acid buffer solution (pH 7.7)and an ethyl acetate solution of 5×10⁻³ mole of 2,4,6-trichlorophenyloxalate (TCPO) and an acetone solution of 5.5×10⁻¹ mole of hydrogenperoxide as the chemical reagents for chemiluminescence were used. Theflow rates of 0.18 ml./min. of the eluting solvent, 0.5 ml./min. of theethyl acetate solution of TCPO, and 1.2 ml./min. of the acetone solutionof hydrogen peroxide were selected by use of the apparatus asillustrated in FIG. 1. The chromatogram obtained is shown in FIG. 2.

In FIG. 2, (1), (2) and (3) are the peaks of dansyl-glutamic acid,dansyl-alanine and dansyl-methionine, respectively.

FIG. 3 shows the calibration curves of the abovementioned threedansylamino acids obtained by the chromatogram in each supplied amount.As is clear from the figure, when the peak height of each dansylaminoacid is taken on the axis of ordinate and the supplied amount of eachdansylamino acid is taken on the axis of abscissa, each calibrationcurve shows good linearity from 8×10⁻¹³ mole to 5×10⁻¹⁴ mole, whichshows the detection limit being 10⁻¹⁴ mole (S/N≈2) as supplied amount.

What is claimed is:
 1. In combination, an analytical apparatus for thequantitative determination of a material selected from the group ofpolycyclic aromatic hydrocarbons, dansylamino acids and vitamins in amixture comprising:(a) means defining and containing a supply of saidmaterials to be separated; (b) a liquid column chromatograph forseparating said material to form a column eluate containing theseparated said material; (c) said material separating means in saidchromatographic column; (d) a first mixing zone; (e) means defining andcontaining a supply of oxalic acid derivative reagent; (f) means forpassing a solution of an oxalic acid derivative to said first mixingzone at a controlled rate; (g) means defining and containing a supply ofhydrogen peroxide reagent; (h) means for passing a solution of hydrogenperoxide to said first mixing zone at a controlled rate; (i) means forsimultaneously introducing the separated solution, oxalic acidderivative reagent and hydrogen peroxide into said first mixing means;(j) means in said first mixing zone to mix said column eluate, saidoxalic acid derivative, and said hydrogen peroxide to form a firstmixture; (k) a coiled mixing zone through which said first mixture iscontinuously passed to form a second mixture, whereby chemiluminescenceoccurs; and (l) a light-free detecting section comprising a flow celland a light receptive detection means for perceiving and multiplying thelight produced by the chemiluminescence reaction.
 2. The apparatus ofclaim 1, wherein said means for defining and containing a supply ofoxalic acid derivative reagent contains oxalic acid derivatives selectedfrom the group consisting of oxalic acid esters, oxalic acid chlorides,oxalic acid anhydrides and oxalic acid amides.